I. Harpin
The Type III Secretion System (TTSS) is an exporting machinery specific for Gram-negative bacteria and is found among plant and animal pathogens, but also in endosymbiotic Rhizobia. TTSS is postulated to deliver proteins into the host cell to which the bacterium is associated. In plant pathogenic bacteria, the TTSS is a cluster of hypersensitive response and pathogenicity genes comprising about 20 genes, the Hrp cluster. Nine of these genes (the harpin conserved or hrc) are conserved among both plant and animal pathogens, eight of them share homology with genes encoding the flagella apparatus (Bogdanove et al., Mol. Microbiol. 20, 681-683, 1996), the ninth, hrcC, is homologous to the GSP outer membrane secretins (Deng and Huang, J. Bacteriol. 180, 4523-4531, 1999). The hpa (hrp-associated) genes contribute to pathogenicity and to the induction of the hypersensitive response (HR) in nonhost plants, but are not essential for the pathogenic interactions of bacteria with plants. The flagella apparatus and the TTSS are postulated to be evolved from a common origin (Gophna et al., Gene 312, 151-163, 2003); the TTSS has furthermore spread among evolutionary distant bacterial species via multiple horizontal-transfer events (Nguyen et al., J. Mol. Microbiol. Biotechnol. 2, 125-144, 2000).
Many gram-negative plant-pathogenic bacteria possess two sets of genes that modulate their interactions with plants. The avirulence genes determine host specificity based on gene—for gene interactions, and the hrp (hypersensitive reaction and pathogenicity) genes are involved in pathogenicity and the induction of hypersensitive responses (HR) in nonhost plants. The HR is a highly localized plant cell death that occurs when non-host plants or resistant cultivars of host plants are infiltrated with the plant pathogen or HR elicitor molecules, such as Avr proteins and harpins. The HR is thought be a resistance reaction of plants to microbial pathogens.
Harpins are a group of HR elicitors that are secreted by the type III secretion pathway (TTSS) and elicit HR when infiltrated into the apoplast of leaves of non-host plants. Unlike Avr proteins, which must be delivered inside the cell to exert their functions, harpins can elicit HR when delivered to the intercellular space of plant cells. Since the first harpin, HrpN, was identified from Erwinia amylovora, many harpins have been reported from various species, including Pseudomonas, Ralstonia, and Xanthomonas. Harpins are glycine-rich, heat stable proteins, lacking cysteine, and are postulated to be present in all plant pathogenic bacteria having a TTSS (Alfano and Colmer, Annu. Rev. Phytopathol. 42, 385-414, 2004). The biochemical mechanism of HR elicitation by harpins in non-host plants remains unclear. HrpZ of Pseudomonas syringae pv. syringae associates with the cell walls rather than the membranes of plant cells, and the protein elicits no response from protoplasts, which lack walls (Hoyos et al. Mol. Plant-Microbe Interact. 9, 608-616, 1996). However, HrpZ of P. syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore (Lee et al., Proc. Natl. Acad. Sci. USA 98, 289-294, 2001). The N-terminal 109 amino acids and the C-terminal 216 amino acids of HrpZ are able to elicit HR to a level similar to full-length HrpZ (Alfano et al., Mol. Microbiol. 19, 715-728, 1996). Kim et al. and Charkowski et al. showed that the HrpW harpins of E. amylovora and P. syringae pv. tomato are composed of two domains—the N-terminal harpin domain and C-terminal Pel (pectate lyase) domain—and proposed that HrpW acts in the cell wall (Charkowski et al., J. Bacteriol. 180, 5211-5217, 1998; Kim and Beer, J. Bacteriol. 180, 5203-5210, 1998).
Besides harpins, the TTSS cluster in bacteria may also include genes encoding Harpin associated Factors. HpaG polypeptides are smaller than harpins, and they share little sequence homology. These sequence differences with harpins are postulated to contribute to the difference in the ability to elicit HR in plants between HpaG polypeptides and harpins (Kim et al., J. Bacteriol. 186, 6239-6247, 2004)
Korean patent application KR20030068302 discloses the Xanthomonas HpaG protein, which, when applied to plants or plant seeds, confers disease resistance, in particular resistance to Xanthomonas axonopodis infection. Harpin associated Factors have been used to confer disease resistance in plants; and as a result of this biotic stress resistance, plants had better yield compared to the control plants under biotic stress conditions.
Surprisingly it has now been found that modulating expression in a plant of a nucleic acid encoding a Harpin-associated Factor G polypeptide (HpaG) give plants enhanced yield-related traits relative to control plant. These enhanced yield-related traits were obtained in plants that were not exposed to stress.
II. SNF2
The present invention concerns a method for enhancing yield-related traits in plants relative to control plants by increasing expression in a plant of a nucleic acid sequence encoding a SWITCH 2/SUCROSE NON-FERMENTING 2 (SWI2/SNF2) polypeptide.
Many chromosome-associated cellular processes, such as replication, transcription, DNA repair, or recombination, require accessible DNA. To deal with these events, cells possess activities that can remodel chromatin in eukaryotes or disrupt other DNA:protein complexes in both pro- and eukaryotes, using ATP hydrolysis. One of the best-studied examples of these activities is carried out by the SWI2/SNF2 family of ATPases, a large group of proteins implicated in many different remodeling-like processes.
SWI2/SNF2 family proteins are ubiquitous, as they are found in bacteria, archaea and eukaryotes. They have recently been classified into 24 distinct subfamilies, after multiple sequence alignment of the SWI2/SNF2 ATPase domain comprising the seven conserved sequence motifs (I, Ia, II, III, IV, V, and VI) (Flaus et al. (2006) Nucleic Acids Res. 2006; 34(10): 2887-2905). These subfamilies have traditionally taken the name of the archetypal member. One subfamily is named SSO1653, after the sole SWI2/SNF2 family member in archaeal Sulfolobus solfataricus (Flaus et al., supra; Duur et al. (2005) Cell 121(3): 363-373), the uniquely archaeal and eubacterial subfamily most similar to the eukaryotic SWI2/SNF2 proteins. The SSO1653 subfamily carries all the SWI2/SNF2 family sequence and structural hallmarks.
US patent application US2003/233670 describes polynucleotides and proteins encoded by the polynucleotides. SEQ ID NO: 125 is a polynucleotide sequence encoding a SWI2/SNF2 polypeptide of the SSO1653 subfamily from Synechocystis sp. PCC 6803. US patent application US2005/108791 describes 24149 nucleic acid and polypeptide sequences, among which a nucleic acid sequence represented by SEQ ID NO: 57 encoding a SWI2/SNF2 polypeptide of the SSO1653 subfamily from Synechocystis sp. PCC 6803, as represented by SEQ ID NO: 396.
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a SWI2/SNF2 polypeptide gives plants having enhanced yield-related traits relative to control plants.